This invention relates generally to the field of viral proteins and more specifically to variants of hepatitis C having truncated E2 proteins.
Hepatitis C Virus (HCV), the etiological agent of post-transfusion and community-acquired non-A, non-B hepatitis is responsible for approximately 20% of all cases of acute hepatitis, 70% of chronic hepatitis and 30% of end-stage liver disease in the United States (Hoofnagle, J. H., Hepatology 26:15S-20S (1997)). Of those chronically infected individuals, 15-30% will develop cirrhosis and 10-30% of these patients will progress to hepatocellular carcinoma and end-stage liver disease. No vaccine is currently available to prevent the disease and treatment options are limited. Development of a safe and efficacious vaccine is hampered by the lack of xe2x80x9ccorrelates of protective immunityxe2x80x9d, the inability to propagate the virus in culture, and the absence of a small animal model.
Based on its genome structure, hepatitis C virus, has been placed in the family Flaviviridae as a separate genus Hepacivirus. The HCV genome is a single positive-stranded RNA of approximately 9,500 nucleotides containing short 5xe2x80x2 and 3xe2x80x2 untranslated regions, and a single long open reading frame. The polyprotein is organized in the order, C (core or nucleocapsid), E1 (envelope1), E2 (E2/NS 1, envelope2), P7 (small protein of unknown function), NS2 (nonstructural), NS3, NS4A, NS4B, NS5A, and NS5B. The mature viral proteins are processed from the polyprotein by co- and post-translational proteolytic cleavage by either viral or host cell proteases.
Phylogenic analysis has identified six distinct HCV genotypes (clades) and multiple minor genetic groups. Infection and recovery from disease caused by one virus genotype does not protect from reinfection by homologous or heterologous virus. Infection and recovery, therefore, does not induce protective immunity.
Vaccine development has focused on two viral structural proteins, E1 and E2 (e.g. U.S. Pat. Nos. 5,942,234; 6,121,020; and 6,150,134). One region of the E2 protein, localized to the N-terminal 30 amino acids (hypervariable region 1 [HVR1]) has been found to be the most heterogeneous region among different virus isolates. Patients with high titer anti-HVR1 antibodies are known to clear the virus, suggesting that there is at least 1 protective epitope within this region, but also suggesting that this region is under direct selective pressure. Anti-HVR1 antibodies are sequence specific, and, therefore, offer no protection from heterologous challenge.
The HCV E2 protein has been shown to bind to the major extracellular loop of CD81, a 25-kDa molecule belonging to the tetraspanin family. The presence of antibodies which inhibit binding to CD81 have been correlated with protection from disease. Epitope mapping studies have suggested that multiple sites within the glycoprotein are responsible for this binding. One epitope lies within the HVR1, while it is clear that a number of others lie outside the HVR1. Monoclonal antibodies isolated from a chronically infected HCV patient were able to inhibit the binding of HCV E2 genotype 1a, 1b, 2a, and 2b recombinant proteins to CD81. These epitopes must lie outside the HVR1 and, therefore, are more conserved across HCV genotypes.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. In particular, HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. Currently, there are 6 distinct, but related genotypes of HCV based on phylogenetic analyses (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88:1711-1715). The polyprotein is processed co- and post-translationally into both structural and non-structural (NS) proteins.
In particular, there are three putative structural proteins, consisting of the N-terminal nucleocapsid protein (termed xe2x80x9ccorexe2x80x9d) and two envelope glycoproteins, xe2x80x9cE1xe2x80x9d (also known as E) and xe2x80x9cE2xe2x80x9d (also known as E2/NS1). (See, Houghton et al., Hepatology (1991) 14:381-388, for a discussion of HCV proteins, including E1 and E2.) E1 is detected as a 32-35 kDa species and is converted into a single endo H-sensitive band of approximately 18 kDa. By contrast, E2 displays a complex pattern upon immunoprecipitation consistent with the generation of multiple species. The HCV envelope glycoproteins E1 and E2 form a stable complex that is co-immunoprecipitable. The HCV E1 and E2 glycoproteins are of considerable interest because they have been shown to be protective in primate studies.
The envelope of the HCV virion remains uncharacterized. Thus, expression studies using recombinant cDNA templates are the only means currently available to study envelope biosynthesis. E1 and E2 are retained within cells and lack complex carbohydrate when expressed stably or in a transient Vaccinia virus system. Since the E1 and E2 proteins are normally membrane-bound in these expression systems, it would be desirable to produce secreted forms to facilitate purification of the proteins for further use and to produce variants that are antigenic and induce a protective immune response.
The present invention is based on the seminal discovery that epitopes outside of HVR1 region of HCV E2 protein are sufficient to induce immune response to HCV and provide immune protection against HCV. The invention provides a recombinant cell system that allows the production of recombinant N- and C-terminally truncated Hepatitis C Virus E2 protein in insect cells. Purification of this extracellularly secreted recombinant protein is described herein. Utilization of this recombinant protein for production of an immunogenic response in vivo to purified E2 protein is also provided herein.
In a first embodiment, the invention provides a secreted polypeptide which is a hepatitis C virus (HCV) E2 polypeptide, lacking all or a portion of its membrane spanning domain such that said E2 polypeptide is capable of secretion into growth medium when expressed recombinantly in a host cell, and further comprising a deletion in at least a portion of its N-terminus ending at about amino acid residue 411, numbered with reference to the HCV E2 amino acid sequence (SEQ ID NO:9).
In a preferred aspect, the secreted polypeptide is a hepatitis C virus (HCV) E2 polypeptide, lacking all or a portion of its membrane spanning domain such that said E2 polypeptide is capable of secretion into growth medium when expressed recombinantly in a host cell, wherein said E2 polypeptide lacks at least a portion of its C-terminus beginning at about amino acid residue 662 and at least a portion of its N-terminus ending at about amino acid residue 411, numbered with reference to the HCV E2 amino acid sequence (SEQ ID NO:9). The invention also provides polynucleotides encoding the polypeptides of the invention. An exemplary polynucleotide is set forth in SEQ ID NO:1.
The invention also provides antibodies that specifically bind to a secreted polypeptide which is a hepatitis C virus (HCV) E2 polypeptide, lacking all or a portion of its membrane spanning domain such that said E2 polypeptide is capable of secretion into growth medium when expressed recombinantly in a host cell, and further comprising a deletion in at least a portion of its N-terminus ending at about amino acid residue 411, numbered with reference to the HCV E2 amino acid sequence (SEQ ID NO:9). An exemplary polypeptide is set forth in SEQ ID NO:8.
In another embodiment, the invention provides a method for preparing an immunogenic composition for treatment of HCV. The method includes (a) forming an immunogenic polypeptide composition comprising a polypeptide of the invention, wherein the immunogenic polypeptide composition is suitable for treating HCV; (b) providing a suitable excipient; and (c) mixing the immunogenic composition of (a) with the excipient of (b).
The invention also includes a method of producing anti-HCV antibodies comprising administering to a mammal an effective amount of an immunogenic polypeptide composition comprising a hepatitis C virus (HCV) E2 polypeptide, lacking all or a portion of its membrane spanning domain such that said E2 polypeptide is capable of secretion into growth medium when expressed recombinantly in a host cell, and further comprising a deletion in at least a portion of its N-terminus ending at about amino acid residue 411, numbered with reference to the HCV E2 amino acid sequence (SEQ ID NO:9).
In one embodiment, the present invention provides an immunogenic polypeptide. The immunogenic polypeptide contains an amino acid sequence wherein the amino acid sequence encodes an HCV E2 polypeptide and is not adjacent to an HCV amino acid sequence naturally adjacent to the HCV E2 polypeptide, wherein the HCV E2 polypeptide does not contain hypervariable region 1 of HCV E2 protein and wherein the immunogenic polypeptide provides immune protection against HCV.
In another embodiment, the present invention provides a method of treating HCV infection. The method comprises administering to a subject having or at risk of having HCV (e.g., a recipient of a blood transfusion or a hospital worker), an effective amount of the immunogenic polypeptide of the present invention or the composition thereof.
In still another embodiment, the present invention provides a method of making an HCV E2 immunogenic polypeptide. The method comprises expressing a polynucleotide sequence encoding the HCV E2 immunogenic polypeptide in an insect cell line.
The invention also provides a method of detecting the presence of HCV in a sample comprising contacting the sample with an antibody of the invention and detecting binding of the antibody to the polypeptide, wherein formation of a complex between the antibody and the E2 polypeptide is indicative of the presence of HCV in the sample. Preferably, the antibody is detectably labeled.